There are certain factors (proteins) present in cells during mitosis that are absent during interphase. The mitotic factors induce premature chromosome condensation in interphase cells and, when injected into Xenopus oocytes, induce meiotic maturation. In this research, we take an immunological approach to probe the nature of the mitotic factors. The specific aims of this work are: (1)\to produce monoclonal antibodies that react specifically with mitotic HeLa cells; and (2)\to provide direct evidence for the role of H[unreadable]1[unreadable] histone phosphorylation in chromosome condensation. We have isolated two hybridoma clones that produce antibodies that react specifically with mitotic and meiotic cells from every species tested as detected by indirect immunofluorescence. The two antibodies, designated MPM-1 and MPM-2, recognize a family of polypeptides with apparent molecular masses of 40 to greater than 200 kilodaltons. Both antibodies reacted strongly with three polypeptide bands of 70, 118, and 182 kilodaltons on polyacrylamide slab gels transferred to nitrocellulose sheets. These bands were found to be phosphoproteins as shown by [unreadable]32[unreadable]P labeling and autoradiography and their removal of alkaline phosphatase treatment. When these antibodies were microinjected, they failed to block the entry of G[unreadable]2[unreadable] cells into mitosis or the meiotic maturation of Xenopus oocytes stimulated by progesterone. However, introduction of these antibodies into mitotic HeLa cells delayed cell division by 3 hrs. The delay appears to be due to transient or partial inhibition of dephosphorylation of proteins that usually occurs during M-G[unreadable]1[unreadable] transition. With regard to our second objective, we observed significant increases in the levels of phosphorylation of histones H[unreadable]1[unreadable] and H[unreadable]3[unreadable] from interphase chromatin undergoing premature chromosome condensation. These data further strengthen the correlation between histone phosphorylation and changes in chromosome condensation associated with the entry of cells into mitosis. (K)